Linking methods, compositions, systems, kits and apparatuses

ABSTRACT

In some embodiments, the disclosure relates generally to methods as well as related compositions, systems, kits and apparatus comprising linking proteins to target compounds and/or to locations of interest using tethers. For example, the tether can be used to link the protein to a target compound, for example, to link an enzyme to a substrate. Similarly, the tether can be used to link the protein at or near a desired location on a surface. In one group of embodiments, the tether includes a polynucleotide and the target compound or location on the surface includes another polynucleotide that is capable of hybridizing to the tether. In such embodiments, the tether can be used to link the protein to the target compound or location using nucleic acid hybridization.

BACKGROUND

The ability of enzymes to catalyze biological reactions is isfundamental to life. A range of biological applications use enzymes tosynthesize various biomolecules in vitro. One particularly useful classof enzymes are the polymerases, which can catalyze the polymerization ofbiomolecules (e.g., nucleotides or amino acids) into biopolymers (e.g.,nucleic acids or peptides). For example, polymerases that can polymerizenucleotides into nucleic acids, particularly in a template-dependentfashion, are useful in recombinant DNA technology and nucleic acidsequencing applications. Many nucleic acid sequencing methods monitornucleotide incorporations during in vitro template-dependent replicationof a target nucleic acid molecule by a polymerase.

When using an enzyme to catalyze a biological reaction of interest, itcan be useful to confine the enzyme so that it is co-localized with itssubstrate. Such co-localization can increase the rate or efficiency ofenzymatic catalysis, thereby increasing the enzymatic activity and/orproduct yield under a given set of reaction conditions. Various methodsof co-localizing enzymes with substrates, typically by immobilizing theenzyme on a support that is then contacted with a solution including thesubstrate, have been reported. However, such methods typically cause areduction in enzyme activity and succeed only at low efficiencies. Suchmethods typically also require modification of the enzyme prior to orafter enzyme immobilization, which can be time-consuming and technicallychallenging to perform. There remains a need in the art for simple,efficient and reliable methods to tether enzymes to, or in the vicinityof, their target substrates so as to increase the rate or product yieldof the enzymatic reaction, as well as more generally to tether proteinsto desired locations.

DETAILED DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D depict exemplary embodiments of steps in constructing atethered polymerase. FIG. 1A depicts a tether structure including apolynucleotide sequences, a primary amine at the 5′ end and a FAM groupat the 3′ end. FIG. 1B depicts the tether structure and SMCC. FIG. 1Cdepicts the tether + SMCC reaction and the resulting product. FIG. 1Ddepicts tethers linked to two cysteine residues of Bst polymerase.

FIG. 2 depicts exemplary results of labeling polymerases with AF647maleimide.

FIG. 3 depicts exemplary results of SMCC activation of FAM-labeledtether oligonucleotides and a maleimide-oligonucleotide-FAM product.

FIG. 4 depicts a tethering reaction and gel analysis of exemplaryreaction products with Klenow fragment (KF) and Bst polymerases.

FIG. 5 depicts an embodiment of a reaction using DTT to reduce disulfidebonds of the polymerase.

FIG. 6 depicts an embodiment of an activation reaction ofFAM-oligonucleotide + SMCC to form a derivatized FAM-oligonucleotideproduct.

FIG. 7 depicts an embodiment of a tethering reaction of a polymerase +derivatized FAM-oligonucleotide tether to form a polymerase linked tooligonucleotide tethers.

FIG. 8 depicts exemplary results of oligonucleotide tethering reactionsperformed with varying salt concentrations.

FIG. 9 depicts exemplary results of sequencing reactions using anuntethered sequencing polymerase.

FIG. 10 depicts exemplary results of sequencing reactions using asequencing polymerase tethered with an oligonucleotide.

DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which these inventions belong. All patents, patentapplications, published applications, treatises and other publicationsreferred to herein, both supra and infra, are incorporated by referencein their entirety. If a definition and/or description is explicitly orimplicitly set forth herein that is contrary to or otherwiseinconsistent with any definition set forth in the patents, patentapplications, published applications, and other publications that areherein incorporated by reference, the definition and/or description setforth herein prevails over the definition that is incorporated byreference.

The practice of the disclosure will employ, unless otherwise indicated,conventional techniques of molecular biology, microbiology andrecombinant DNA techniques, which are within the skill of the art. Suchtechniques are explained fully in the literature. See, for example,Sambrook, J., and Russell, D. W., 2001, Molecular Cloning: A LaboratoryManual, Third Edition; Ausubel, F. M., et al., eds., 2002, ShortProtocols In Molecular Biology, Fifth Edition.

As used herein, the terms “a,” “an,” and “the” and similar referentsused herein are to be construed to cover both the singular and theplural unless their usage in context indicates otherwise. Accordingly,the use of the word “a” or “an” when used in the claims orspecification, including when used in conjunction with the term“comprising”, may mean “one,” but it is also consistent with the meaningof “one or more,” “at least one,” and “one or more than one.”

As used herein, the terms “link”, “linked”, “linkage” and variantsthereof comprise any type of fusion, bond, adherence or association thatis of sufficient stability to withstand use in the particular biologicalapplication of interest. Such linkage can comprise, for example,covalent, ionic, hydrogen, dipole-dipole, hydrophilic, hydrophobic, oraffinity bonding, bonds or associations involving van der Waals forces,mechanical bonding, and the like. Optionally, such linkage can occurbetween a combination of different molecules, including but not limitedto: between a nanoparticle and a protein; between a protein and a label;between a linker and a functionalized nanoparticle; between a linker anda protein; between a nucleotide and a label; and the like. Some examplesof linkages can be found, for example, in Hermanson, G., BioconjugateTechniques, Second Edition (2008); Aslam, M., Dent, A., Bioconjugation:Protein Coupling Techniques for the Biomedical Sciences, London:Macmillan (1998); Aslam, M., Dent, A., Bioconjugation: Protein CouplingTechniques for the Biomedical Sciences, London: Macmillan (1998).

In some embodiments, the disclosure relates generally to methods (aswell as related compositions, systems, kits and apparatus) comprisinglink proteins to target compounds and/or to locations of interest usingtethers. For example, the tether can be used to link the protein to atarget compound, for example, to link an enzyme to a substrate.Similarly, the tether can be used to link the protein at or near adesired location on a surface. In one group of embodiments, the tetherincludes a polynucleotide and the target compound or location on thesurface includes another polynucleotide that is capable of hybridizingto the tether. In such embodiments, the tether can be used to link theprotein to the target compound or location using nucleic acidhybridization.

Linking proteins to desired targets or locations by exploiting theability of nucleic acid molecules to selectively hybridize to each otherhas several advantages. For example, methods of affixing nucleic acidsto surfaces, optionally in array format, are well-developed and caneasily be adapted for use in protein-based assays. Similarly, the use ofnucleic acid hybridization as a tethering mechanism is simple toperform, can be reversed at will via appropriate adjustment of reactionconditions, can be employed using native (i.e., unmodified) nucleic acidmolecules and eliminates the need to use binding catalysts or otherreactants. Furthermore, by exploiting the ability of polynucleotides tohybridize with each other in a sequence-specific manner, reactions canbe performed in multiplex format where different groups of protein areselectively linked to different targets or locations using the samelinking conditions.

The use of tethering mechanisms can also be useful in decreasing thecost or effort associated with perfoming protein-based applications. Forexample, many such applications (e.g., enzyme reactions) can consumelarge amounts of protein, which can be costly and time-consuming toprepare. This problem is aggravated when using proteins in methods thatmonitor and detect aggregate signals from a population of proteinmolecules acting upon a population of targets, either in asynchronous(e.g., single molecule) or synchronous format. This problem can beespecially intractable when multiple rounds of washing or reagentexchange are involved. In such methods, it can be very costly torepeatedly provide proteins for each fresh round of reaction,particularly at sufficiently high concentrations to permit reaction ofthe protein with a target. In such situations, the use of tethers tolink the protein to the target or to a surface can eliminate loss ofproteins during reaction washes, thereby reducing or eliminating theneed to replenish the protein in the reaction following each wash. Forexample, multiple reaction and wash cycles can be performed withoutconsuming large amounts of expensive protein reagents in each wash.

Tethering can also effectively increase the local concentration of theprotein within the zone of reaction with a target, thus effectivelyincrease the rate of reaction and/or increasing the total amount ofproduct formed within a given amount of time. For example, tethering ofan enzyme can increase the rate of an enzyme-catalyzed reaction.Typically, such rate is limited by enzyme concentration; tetheringlimits the ability of the enzyme to diffuse away from the reaction site,effectively increasing the localized protein concentration withoutrequiring the use of very large amounts of protein.

Finally, use of tethered proteins can sometimes increase the spectrum ofreaction conditions available to the user. For example, proteintethering can permit the use of reaction conditions that enhance proteinactivity but would otherwise cause loss of untethered proteins from thereaction mixture. Described further herein are some exemplaryembodiments that further illustrate the various advantages of tetheredproteins. For example, use of a tethered polymerase in nucleotideincorporation and/or primer extension applications, such as cyclical(“step-wise”) sequencing-by-synthesis reactions, can reduce the amountof polymerase consumed in each extension step, can increase the reactionrate and/or the total amount of product formed under given reactionconditions and can also permit the use of high-salt reaction conditionswithout significant loss of polymerase between washes, thus effectivelyincreasing the amount of signal obtained from each extension andreducing the amount of incomplete extensions at each step.

In some embodiments, the disclosure relates generally to methods,compositions, systems, apparatuses and kits for linking a protein to asurface, comprising: contacting a tethered protein with a surface, wherethe tethered protein includes a tether linked to protein, and where thetether of the tethered protein includes a surface-reactive moiety andthe surface includes a tether-reactive moiety that is capable ofreacting with the surface-reactive moiety; and forming a linkage betweenthe surface-reactive moiety with the tether-reactive moiety, therebylinking the tethered protein to the surface. In some embodiments, thetether reactive moiety and the surface-reactive moiety each comprise oneof two complementary members of a binding pair. The binding pair can beselected from a group consisting of: a biotin moiety and an avidinmoiety, an antigenic epitope and an antibody or immunogically reactivefragment thereof, an antibody and a hapten, a digoxigen moiety and ananti-digoxigen antibody, a fluorescein moiety and an anti-fluoresceinantibody, an operator and a repressor, a nuclease and a nucleotide, alectin and a polysaccharide, a steroid and a steroid-binding protein, anactive compound and an active compound receptor, a hormone and a hormonereceptor, an enzyme and a substrate, an immunoglobulin and protein A,and two polynucleotides that are complementary to each other over atleast some portion of their respective lengths (where complementarity isoptionally defined according to conventional Watson-Crick base pairingrules or alternatively according to some other base-pairing paradigm).

In some embodiments, the surface-reactive moiety of the tether includesa first polynucleotide having a first polynucleotide sequence, and thetether-reactive moiety of the surface includes a second polynucleotidehaving a second polynucleotide sequence, where the first and secondpolynucleotide sequences are at least 80% complementary to each other(where complementarity is optionally defined according to conventionalWatson-Crick base pairing rules or alternatively according to some otherbase-pairing paradigm). In some embodiments, the first and secondpolynucleotide sequences are at least 85%, at least 90%, at least 95%,at least 97% or at least 99% complementary to each other.

In some embodiments, the disclosure relates generally to methods (andrelated compositions, systems, apparatuses and kits) for linking aprotein to a surface, comprising: binding a tethered protein to asurface, where the tethered protein includes a protein linked to atether, the tether including a first polynucleotide and the firstpolynucleotide including a first polynucleotide sequence; where thebinding further includes contacting the tethered protein with a secondpolynucleotide, wherein the second polynucleotide is linked to a surfaceand includes a second polynucleotide sequence that is at least 80%complementary to the first polynucleotide sequence, and hybridizing thefirst polynucleotide sequence to the second polynucleotide sequence,thereby linking the tethered polymerase to the surface. In someembodiments, the first and second polynucleotide sequences are at least80% complementary to each other (where complementarity is optionallydefined according to conventional Watson-Crick base pairing rules oralternatively according to some other base-pairing paradigm). In someembodiments, the first and second polynucleotide sequences are at least85%, at least 90%, at least 95%, at least 97% or at least 99%complementary to each other.

In some embodiments, the disclosure relates generally to methods,compositions, systems, apparatuses and kits useful for co-localizing anenzyme and its substrate using a tether to link the enzyme to, or in thevicinity of, the substrate. In some embodiments, the colocalization canbe performed prior to, or during, the reaction of the enzyme with thesubstrate. Such co-localization can increase the rate of the enzymaticreaction and/or increase the the product yield.

In some embodiments, the disclosure relates generally to methods ofco-localizing an enzyme with a substrate by linking an enzyme (or anybiologically active fragment thereof) to a substrate using a tether,thereby forming an tethered enzyme-substrate complex that includes theenzyme (or biologically active fragment) linked to the substrate throughthe tether. Typically, the linking is done in such a manner that theenzyme (or biologically active fragment) retains enzymatic activity andcan still react with the substrate to form a product after the linkingis complete. In one exemplary embodiment, the enzyme-reactive moiety ofthe tether can be linked to the enzyme (or biologically active fragment)to form a tethered enzyme. The tethered enzyme can be contacted with thesubstrate under conditions where the substrate-reactive moiety of thetether in the tethered enzyme binds to the substrate, thereby forming atethered enzyme-substrate complex that includes the enzyme linked to thesubstrate through the tether. Optionally, the tethered enzyme-substratecomplex retains enzymatic activity. For example, the enzyme (orbiologically active fragment) of the tethered enzyme-substrate complexcan bind to the substrate within the tethered enzyme-substrate complex,or to any other substrate molecule within the reaction mixture, and cancatalyze the enzyme-substrate reaction. In some embodiments, thesubstrate is linked to a surface, such that formation of the tetheredenzyme-substrate complex effectively localizes the enzyme (orbiologically active fragment) to the surface.

In some embodiments, the tether is not used to link the enzyme to thesubstrate, but is instead used to link the enzyme to a surface. Forexample, the tether can link the enzyme to to the surface, therebyforming a surface-linked tethered enzyme, while the substrate can befree-floating or independently attached to the same or differentsurface. In some embodiments, the substrate is linked to the samesurface as the the tethered enzyme. For example, the enzyme andsubstrate can each be independently linked to the same surface atattachment sites that are sufficiently close to each other, and wherethe tether is sufficiently flexible, to permit reaction of the enzymewith the substrate.

In some embodiments, the disclosure relates generally to methods (andrelated compositions, systems, kits and apparatuses) for co-localizingan enzyme and its substrate comprising: forming a tethered enzyme bylinking a tether to the enzyme (or biologically active fragmentthereof), where the tethered enzyme retains enzymatic activity; andbinding the tethered enzyme to a substrate. Optionally, the tetherincludes an enzyme-reactive moiety, and the linking further includesreacting the enzyme-reactive moiety with the enzyme, thereby forming anenzyme-tether linkage that links the tether to the enzyme. The linkagecan be a covalent linkage, an electrostatic linkage or any other linkagesuitable for linking the enzyme (or biologically active fragment) to thetether. In some embodiments, the tether includes a substrate-reactivemoiety, and the disclosed methods (and related compositions, systems,kits and apparatuses) further involve linking the tether to thesubstrate by reacting the substrate-reactive moiety with the substrate,and the linking further includes reacting the substrate-reactive moietywith the substrate, thereby forming an substrate-tether linkage thatlinks the tether to the substrate. In some embodiments, the enzyme inthe tethered enzyme selected from the group consisting of: a polymerase,a ribosome, a helicase, a pyrophosphatase and an apyrase.

In some embodiments, the disclosure relates generally to methods (andrelated compositions, systems, apparatuses and kits) for linkingpolymerases to target compounds, or to desired locations of interest. Insome embodiments, the polymerase can include that is capable ofcatalyzing the incorporation of one or more nucleotides into a nucleicacid molecule. Typically but not necessarily such nucleotideincorporation can occur in a template-dependent fashion. The polymerasecan be a naturally-occurring polymerase, or any subunit or truncationthereof, a mutant, variant, derivative, recombinant, fusion, modified,chemically modified, or otherwise engineered form of any polymerase, asynthetic molecule or assembly that can catalyze nucleotideincorporation, as well as analogs, derivatives or fragments thereof thatretain the ability to catalyze such nucleotide incorporation.Optionally, the polymerase can be a mutant polymerase comprising one ormore mutations involving the replacement of one or more amino acids withother amino acids, the insertion or deletion of one or more amino acids,or the linkage of parts of two or more polymerases. Typically, thepolymerase comprises one or more active sites at which nucleotidebinding and/or catalysis of nucleotide polymerization can occur. Someexemplary polymerases include without limitation DNA polymerasesincluding both DNA-dependent and RNA-dependent DNA polymerases (such asfor example Bst DNA polymerase, Therminator™ polymerase, KOD polymerase,Phi-29 DNA polymerase, reverse transcriptases and E. coli DNApolymerase) and RNA polymerases. In some embodiments, the polymerase isa fusion proteins comprising at least two portions linked to each other,where the first portion comprises a peptide that can catalyze thepolymerization of nucleotides into a nucleic acid strand and is linkedto a second portion that comprises a second polypeptide, such as, forexample, a reporter enzyme or a processivity-enhancing domain. In oneexemplary embodiment, the polymerase is Phusion® DNA polymerase (NewEngland Biolabs), which comprises a Pyrococcus-like polymerase fused toa processivity-enhancing domain as described, for example, in U.S. Pat.No. 6,627,424.

Typically, the polymerase includes a nucleic acid binding site and apolymerase active site. The polymerase active site can be a site ofpolymerase activity. The polymerase activity can comprise any in vivo orin vitro enzymatic activity of the polymerase that relates to catalyzingthe incorporation of one or more nucleotides into a nucleic acidmolecule, for example, primer extension activity and the like.Typically, but not necessarily such nucleotide polymerization occurs ina template-dependent fashion. In addition to such polymerase activity,the polymerase can possess other activities such as DNA bindingactivity, 3′ to 5′ or 5′ to 3′ exonuclease activity, and the like.

In some embodiments, the disclosure relates generally to methods (andrelated compositions, systems, kits and apparatuses) for linking apolymerase to a surface, comprising: contacting a tethered polymerasewith a surface, where the tethered polymerase includes polymerase linkedto a tether including a first polynucleotide, where the polynucleotidehas a first polynucleotide sequence, and the surface includes a secondpolynucleotide including a second polynucleotide sequence, where thefirst and second polynucleotide sequences are at least 80% complementaryto each other; and hybridizing the first polynucleotide sequence to thesecond polynucleotide sequence, thereby linking the tethered polymeraseto the surface.

In some embodiments, the first polynucleotide of the tether iscovalently linked to an amino acid residue of the polymerase. Forexample, the first polynucleotide can be covalently linked to a cysteineresidue of the polymerase. The first polynucleotide can be covalentlylinked to the cysteine residue through a flexible linker.

Typically, the tethered polymerase has polymerase activity, and cancatalyze the incorporation of one or more nucleotides into a nucleicacid molecule.

In some embodiments, the disclosed methods (and related compositions,systems, kits and apparatuses) can further include contacting thetethered polymerase with one or more nucleotides.

In some embodiments, the disclosed methods (and related compositions,systems, kits and apparatuses) can further include incorporating one ormore nucleotides into the second polynucleotide using the tetheredpolymerase. For example, the tethered polymerase can polymerize the oneor more nucleotides onto the 3′ end of the second polynucleotide.

In some embodiments, the surface includes a third polynucleotide, andthe disclosed methods (and related compositions, systems, kits andapparatuses) can further include incorporating one or more nucleotidesinto the third polynucleotide using the tethered polymerase. Forexample, the tethered polymerase can polymerize the one or morenucleotides onto the 3′ end of the third polynucleotide.

In some embodiments, the disclosure relates generally to compositions(as well as related methods, systems, kits and apparatus) comprisingtethered proteins, where the tether can be fixed to a site of interest.For example, the tether can link the protein to a substrate or to asurface.

In some embodiments, the disclosure relates generally to compositions(and related methods, systems, kits and apparatuses) comprising tetheredenzymes. Typically, the tethered enzyme includes an enzyme linked to atether, where the tethered enzyme has enzymatic activity.

In some embodiments, the tether is a linear tether. In some embodiments,the tether can include a linear polymer, which can be rigid or flexible.In some embodiments, the tether includes a polynucleotide. In someembodiments, the tether includes a flexible linker. In some embodiments,the flexible linker can have a persistence length of up to about 200 nm;for example between about 0.5 nm and about 1500 nm; typically betweenabout 1.0 nm and about 15 nm or between about 1.5 nm and about 10 nm;even more typically between about 1.5 nm and 5.0 nm, where thepersistence length is measured under any reaction conditions that aresuitable for primer extension (for example, in a buffer comprisingbetween 0 and about 2M salt (e.g., NaCl or MgCl₂)), even more typicallyin a buffer comprising between 0 and about 100 mM salt (e.g., NaCl orMgCl₂). Methods of measuring persistence length of polynucleotides insolution are known in the art; see, e.g., Murphy et al., “Probing SingleStranded DNA Conformational Flexibility Using FluorescenceSpectroscopy”, Biophys J. 2004 April; 86(4): 2530-2537. In someembodiments, the flexible linker includes a polynucleotide. In someembodiments, the flexible linker includes a polypeptide. In someembodiments, the flexible linker includes polyethylene glycol.

Optionally, the tether can include one or more labels. The one or morelabels can be detected using any suitable detection system. For examplethe one or more labels can include fluorescent labels, luminescentlabels, chemically detectable labels, or magnetically detectable labels.

In some embodiments, the tether can include one or more reactivemoieties. In some embodiments, the tether includes an enzyme-reactivemoiety. The enzyme-reactive moiety can react with the enzyme undersuitable conditions. Reaction of the enzyme-reactive moiety with theenzyme can result in the formation of an enzyme-tether linkage thatlinks the tether to the enzyme, thereby forming a tethered enzyme. Theenzyme-tether linkage can include one or more bonds selected from thegroup consisting of: a covalent bond, an electrostatic bond, anaffinity-based interaction and a hydrogen bond. In some embodiments, theenzyme-reactive moiety of the tether reacts with a sulfhydryl group of acysteine residue of the enzyme and forms a covalent bond with a sulfuratom of the sulfhydryl group of the cysteine. In some embodiments, theenzyme-reactive moiety of the tether reacts with an amino group of anamino acid residue of the enzyme. The amino group can be located on anN-terminal amino acid residue of the enzyme. In some embodiments, theenzyme-reactive moiety of the tether reacts with a carboxyl group of anamino acid residue of the enzyme. The carboxyl group can be located on aC-terminal amino acid residue of the enzyme.

In some embodiments, the enzyme includes a reactive sulfhydryl group(for example, a sulfhydryl group of a cysteine residue, that isoptionally a surface cysteine) and the enzyme-reactive moiety of thetether includes a linking group that is capable of reacting with thesulfhydryl group of the cysteine reside. For example, the linking groupcan include a reactive amine that can be activated by suitable treatment(e.g., reaction with SMCC) to form a reactive maleimide. The tetherincluding the reactive maleimide can then be contacted with the enzymeunder suitable conditions where the reactive maleimide group reacts withthe sulfhydryl group of the cysteine, forming a covalent linkage,typically a linkage comprising a thioether bond.

In some embodiments, the tether includes a substrate-reactive moiety.The substrate-reactive moiety can react with the substrate undersuitable conditions. For example, the substrate-reactive moiety canreact or bind selectively to the substrate when contacted with thesubstrate Reaction of the substrate-reactive moiety with the substratecan result in the formation of a substrate-tether linkage that links thetether to the substrate. The substrate-tether linkage can include one ormore bonds selected from the group consisting of: a covalent bond, anelectrostatic bond, an affinity-based interaction and a hydrogen bond.In one exemplary embodiment, the substrate-reactive moiety of the tethercomprises a first polynucleotide including a first polynucleotidesequence, and the substrate includes, e.g., is linked to, a secondpolynucleotide including a second polynucleotide sequence, where thefirst and second polynucleotide sequences are at least 80% complementaryto each other. When the tether and substrate are contacted with eachother under hybridization conditions, then first and secondpolynucleotide sequences hybridize to each other, thereby forming anucleic acid duplex that links the tether to the substrate.

In some embodiments, the tether of the tethered enzyme includes asurface-reactive moiety. The surface-reactive moiety can react with asurface under suitable conditions. Reaction of the surface-reactivemoiety with the surface can result in the formation of a surface-tetherlinkage that links the tether to the surface. The surface-tether linkagecan include one or more bonds selected from the group consisting of: acovalent bond, an electrostatic bond, an affinity-based interaction anda hydrogen bond.

In some embodiments, the surface-reactive moiety of the tethered enzymeincludes a first polynucleotide, and the surface includes a secondpolynucleotide. The first and second polynucleotides may be capable ofhybridizing to each other over at least a portion of their respectivelengths. In some embodiments, the first polynucleotide includes a firstpolynucleotide sequence and the second polynucleotide includes a secondpolynucleotide sequence, where the first and second polynucleotidesequences are at least 80% complementary to each other. In someembodiments, the first and second polynucleotide sequences are at least80% complementary, at least 90% complementary, at least 95%complementary, at least 97% complementary, or at least 99% complementaryto each other. Typically, complementarity can be defined according toconventional Watson-Crick base pairing rules (e.g., A base pairs with Tand C base pairs with G); alternatively complementarity can be definedaccording to non-Watson Crick base pairing paradigms as well. In someembodiments, the first polynucleotide sequence and the secondpolynucleotide sequence are hybridized to each other.

In some embodiments, the tether is linked to any one or more members ofthe group consisting of: enzyme, substrate and surface. For example, thetether can first be linked to the enzyme through the enzyme-reactivemoiety of the tether, thereby forming a tethered enzyme, and the tetherof the tethered enzyme can then be linked to a substrate using thesubstrate-reactive moiety of the tether, or alternatively linkeddirectly to a surface using a surface-reactive moiety of the tether.Alternatively, the tether can first be linked to a substrate, or to asurface, and then be linked to the enzyme.

In a typical embodiment, the enzyme comprises a polymerase.

In some embodiments, the disclosure relates generally to a compositioncomprising a tethered polymerase including a polymerase linked to atether, where the tethered polymerase has polymerase activity. Thepolymerase-tether linkage can include one or more bonds selected fromthe group consisting of: a covalent bond, an electrostatic bond, anaffinity-based interaction and a hydrogen bond. In some embodiments, thepolymerase can be covalently linked to the tether. The tether can belinked to an amino acid residue of the polymerase. In some embodiments,the tether is linked to a sulfhydryl group of a cysteine residue of thepolymerase. In some embodiments, the tether is linked to an amino groupof an amino acid residue of the polymerase. In some embodiments, thetether is linked to a carboxyl group of an amino acid residue of thepolymerase. The tether can include a polynucleotide. The polymerase canbe covalently linked to the polynucleotide. The polynucleotide can becovalently linked to a cysteine residue of the polymerase. In someembodiments, a 5′ end of the polynucleotide is covalently linked to anamino acid residue of the polymerase. The covalent linkage between thetether and the polymerase can include a series of atoms, each atom inthe series being linked covalently to the next atom in the series suchthat the tether and the polymerase are ultimately linked to each otherthrough a series of atoms linked through covalent bonds.

In some embodiments, the polymerase of the tethered polymerase is linkedto a first member of a binding pair, the tether is linked to a secondmember of the binding pair, and where the first member and the secondmember are linked to each other to form the tethered polymerase. Thebinding pair can be selected from a group consisting of: a biotin moietyand an avidin moiety, an antigenic epitope and an antibody orimmunogically reactive fragment thereof, an antibody and a hapten, adigoxigen moiety and an anti-digoxigen antibody, a fluorescein moietyand an anti-fluorescein antibody, an operator and a repressor, anuclease and a nucleotide, a lectin and a polysaccharide, a steroid anda steroid-binding protein, an active compound and an active compoundreceptor, a hormone and a hormone receptor, an enzyme and a substrate,an immunoglobulin and protein A, and two polynucleotides that arecomplementary to each other over at least some portion of theirrespective lengths (where complementarity can optionally be definedaccording to conventional Watson-Crick base pairing rules oralternatively according to some other base-pairing paradigm).

As used herein, the term “nucleotide” and its variants comprises anycompound that can bind selectively to, or can be polymerized by, apolymerase. Typically, but not necessarily, selective binding of thenucleotide to the polymerase is followed by polymerization of thenucleotide into a nucleic acid strand by the polymerase; occasionallyhowever the nucleotide may dissociate from the polymerase withoutbecoming incorporated into the nucleic acid strand, an event referred toherein as a “non-productive” event. Such nucleotides include not onlynaturally-occurring nucleotides but also any analogs, regardless oftheir structure, that can bind selectively to, or can be polymerized by,a polymerase. While naturally-occurring nucleotides typically comprisebase, sugar and phosphate moieties, the nucleotides of the disclosurecan include compounds lacking any one, some or all of such moieties. Insome embodiments, the nucleotide can optionally include a chain ofphosphorus atoms comprising three, four, five, six, seven, eight, nine,ten or more phosphorus atoms. In some embodiments, the phosphorus chaincan be attached to any carbon of a sugar ring, such as the 5′ carbon.The phosphorus chain can be linked to the sugar with an intervening O orS. In one embodiment, one or more phosphorus atoms in the chain can bepart of a phosphate group having P and O. In another embodiment, thephosphorus atoms in the chain can be linked together with intervening O,NH, S, methylene, substituted methylene, ethylene, substituted ethylene,CNH₂, C(O), C(CH₂), CH₂CH₂, or C(OH)CH₂R (where R can be a 4-pyridine or1-imidazole). In one embodiment, the phosphorus atoms in the chain canhave side groups having O, BH₃, or S. In the phosphorus chain, aphosphorus atom with a side group other than O can be a substitutedphosphate group. Some examples of nucleotide analogs are described inXu, U.S. Pat. No. 7,405,281. In some embodiments, the nucleotidecomprises a label (e.g., reporter moiety) and referred to herein as a“labeled nucleotide”; the label of the labeled nucleotide is referred toherein as a “nucleotide label”. In some embodiments, the label can be inthe form of a fluorescent dye attached to the terminal phosphate group,i.e., the phosphate group or substitute phosphate group most distal fromthe sugar. Some examples of nucleotides that can be used in thedisclosed methods and compositions include, but are not limited to,ribonucleotides, deoxyribonucleotides, modified ribonucleotides,modified deoxyribonucleotides, ribonucleotide polyphosphates,deoxyribonucleotide polyphosphates, modified ribonucleotidepolyphosphates, modified deoxyribonucleotide polyphosphates, peptidenucleotides, metallonucleosides, phosphonate nucleosides, and modifiedphosphate-sugar backbone nucleotides, analogs, derivatives, or variantsof the foregoing compounds, and the like. In some embodiments, thenucleotide can comprise non-oxygen moieties such as, for example, thio-or borano-moieties, in place of the oxygen moiety bridging the alphaphosphate and the sugar of the nucleotide, or the alpha and betaphosphates of the nucleotide, or the beta and gamma phosphates of thenucleotide, or between any other two phosphates of the nucleotide, orany combination thereof.

As used herein, the term “nucleotide incorporation” and its variantscomprise polymerization of one or more nucleotides to form a nucleicacid strand including at least two nucleotides linked to each other,typically but not necessarily via phosphodiester bonds, althoughalternative linkages may be possible in the context of particularnucleotide analogs.

The following non-limiting examples are provided purely by way ofillustration of exemplary embodiments, and in no way limit the scope andspirit of the present disclosure. Furthermore, it is to be understoodthat any inventions disclosed or claimed herein encompass allvariations, combinations, and permutations of any one or more featuresdescribed herein. Any one or more features may be explicitly excludedfrom the claims even if the specific exclusion is not set forthexplicitly herein. It should also be understood that disclosure of areagent for use in a method is intended to be synonymous with (andprovide support for) that method involving the use of that reagent,according either to the specific methods disclosed herein, or othermethods known in the art unless one of ordinary skill in the art wouldunderstand otherwise. In addition, where the specification and/or claimsdisclose a method, any one or more of the reagents disclosed herein maybe used in the method, unless one of ordinary skill in the art wouldunderstand otherwise.

EXAMPLES Example 1: Construction of a Tethered Polymerase

A tethered polymerase was constructed by covalently linking a tetherincluding a first polynucleotide sequence to a cysteine residue of avariant Bst polymerase (“Ion Sequencing Polymerase”) having thefollowing amino acid sequence:

MAKMAFTLADRVTEEMLADKAALVVEVVEENYHDAPIVGIAVVNERGRFFLRPETALADPQFVAWLGDETKKKSMFDSKRAAVALKWKGIELCGVSFDLLLAAYLLDPAQGVDDVAAAAKMKQYEAVRPDEAVYGKGAKRAVPDEPVLAEHLVRKAAAIWELERPFLDELRRNEQDRLLVELEQPLSSILAEMEFAGVKVDTKRLEQMGKELAEQLGTVEQRIYELAGQEFNINSPKQLGVILFEKLQLPVLKKTKTGYSTSADVLEKLAPYHEIVENILHYRQLGKLQSTYIEGLLKVVRPDTKKVHTIFNQALTQTGRLSSTEPNLQNIPIRLEEGRKIRQAFVPSESDWLIFAADYSQIELRVLAHIAEDDNLMEAFRRDLDIHTKTAMDIFQVSEDEVTPNMRRQAKAVNFGIVYGISDYGLAQNLNISRKEAAEFIERYFQSFPGVKRYMENIVQEAKQKGYVTTLLHRRRYLPDITSRNFNVRSFAERMAMNTPIQGSAADIIKKAMIDLNARLKEERLQAHLLLQVHDELILEAPKEEMERLCRLVPEVMEQAV TLRVPLKVDYRYGSTWYDAK

The tether included a polynucleotide sequence complementary to thesequence of a sequencing primer P1 (described further below) and aflexible linker including a poly-A stretch, as well as a primary amineat the 5′ end of tether. The 3′ end of the tether was blocked using aFAM group. The structure of the tether is depicted in FIG. 1A.

The primary amine of the tether can be activated via conversion to amaleimide group using SMCC, as shown in FIG. 1B. The reaction andresulting product is depicted in FIG. 1C.

The tether of FIG. 1 was then covalently linked to two cysteine residuesof the Bst Polymerase to obtain the tethered polymerase depicted in FIG.1D according to the methods described below.

Activation of the Tether

The following reagents were used:

The tether including a 49 base-pair oligonucleotide with a tB30 aminotether and FAM; MW=16115.9; 105.7 nm=1.7 mg (IDT DNA). The structure andprimary amino acid sequence of the tether can be depicted as follows:

5AmMC12/AA AAA AAA AAA AAA AAA AAA GAC TGCCAA GGC ACA CAG GGG ATA GGA AA/36-FAM

Bst; Ion Torrent lot 7 sequencing polymerase, ˜1 mg/mL

Klenow (KF) exo-wt (106.9 μM; 7.6 mg/mL; Starlight source)

AlexaFluor 647 maleimide (1 mg; A20347; MW˜1300)

AlexaFluor 647 cadaverine (1 mg; A30679; MW˜1000)

SMCC (Molecular Probes; 51534; Lot: 38985A; MW=334.33)

sulfo-SMCC (Molecular Biosciences; MW=463.37)

sodium bicarbonate (J.T. BAKER; 3506-1; FW 84.01)

The Bst polymerase was then labeled with maleimide dye (AF647maleimide). Results are depicted in FIG. 2.

The tether including the FAM-labeled oligonucleotide was activated withSMCC to generate a maleimide-oligo-FAM product. Representative resultsof the activation reaction are depicted in FIG. 3.

The maleimide-oligo-FAM product was purified with NAP-5 column

Two different polymerases, Klenow fragment (“KF”) and the variant Bstpolymerase (“Bst”) were reacted with maleimide-oligo-FAM to formtethered polymerases, and the reaction products were purified on a gel.Representative results are depicted in FIG. 4. A possible product bandwas detected using gel analysis using less than 10% of the reaction.

Linkage of the Polymerase to the Tether

The following protocol was used to link the polymerase to the tether:

(a) Polymerase DTT Treatment and Purification to Reduce Disulfide Bonds:

Reagents (Stock Concentration) and Materials:

Lot 7 sequencing Bst polymerase (˜1 mg/mL), DTT (1 M),

Tris pH 8.5 (1 M), Exchange Buffer (50 mM Mes pH 6.0, 2 mM EDTA) NAP-5column (G.E. NAP column, product#17-0853-02),

Amicon 30K centrifugal filter (Millipore Cat No. UFC503096 Lot No.ROMA72710), 1-1.5 mL Eppendorf Tubes

Protocol:

Mix Bst polymerase (25 μL), DTT (1 μL), Tris pH 8.5 (5 μL) and ddH₂O (69μL). Incubate at 4° C. for 1-12 h. Purify Bst polymerase using a NAP-5column Use gravity flow to prepare and use the NAP-5 column: (1) drainstorage buffer (2) add exchange buffer and pass >15 mL exchange buffer(3) add reaction mix and drain until all of the mix has entered thematrix and (4) fill column with exchange buffer and (6) collect 5 dropfractions. Next use the Nanodrop to determine in which fraction(s)contain the Bst polymerase. Pool the fractions and concentrate using theAmicon 30K centrifugal filter using the manufacture's instructions.

(Amicon Protocol: spin for 7 min at 11,000×g. Transfer filter to cleancollection tube and insert in an inverted manner. Spin for 1 mM at1000×g). Measure the absorbance of the concentated Bst polymerase usingthe Nanodrop and determine the Bst concentration using the followingformula:ε₂₈₀=58000/Mcm.

The reaction is depicted in FIG. 5.

(b) Activation of FAM-Oligo with SMCC

The oligo tether was activated using SMCC to generate a FAM-oligoderivatized with maleimide

Reagents:

SMCC (MW=334.33 g/mol; Molecular Probes; product #:S1534; Lot:38985A),

Sodium bicarbonate (J.T. Baker; product #:3506-01)

FAM-oligo (tB30 Amino tether FAM; MW=16115.9; 105.7 nm=1.7 mg),

DMSO

Protocol:

FAM-oligo was dissolved in ddH₂O to obtain a 500 μM concentration.Approximately 1 mg of SMCC was weighed and dissolved with DMSO toprepare a 20 mM concentration. The following reaction mix was prepared:FAM-oligo (20 μL), NaHCO3 (20 μL) and SMCC (10 μL). The reaction wasincubated at room temperature for 20-30 mM The product was purifiedusing a NAP-5 column. The same protocol to purify the polymerase wasused to purify the product. The fractions were identified with UV-Vis(nanodrop). An aliquot of the identified fraction was diluted with anequal amount of 250 mM phosphate (pH 9) to measure the absorbance of FAMand use a molar extinction coefficient of ε₅₁₀=58000/Mcm to determinethe FAM concentration. Analytical HPLC confirmed that the desiredproduct. However, an additional peak was also observed. Thiscorresponding peak is probably the product with a hydrolyzed maleimide.Reducing the reaction pH and time minimizes the production of thisproduct.

The reaction is depicted in FIG. 6.

(c) Linking the Tether to the Polymerase

The tether was linked to the polymerase to form a tethered polymerase asfollows:

Reagents:

Bst (DTT treated and purified),

FAM-oligo-Maleimide (freshly prepared)

Exchange buffer (50 mM MES pH 6.0, 2 mM EDTA or 50 mM ACES pH 6.8, 2 mMEDTA)

NaCl

Protocol:

Mix Bst and FAM-oligo-maleimide in exchange buffer in the presence of0.5-1 M NaCl.

Incubate reaction at 4° C. for ˜12 h

The reaction is depicted in FIG. 7.

As depicted in FIG. 8, the use of varying salt concentrations in theexchange buffer resulted in different product yields, with the inclusionof NaCl pushing the reaction yield to greater than 50%.

Example 2: Use of Tethered Polymerase for Nucleic Acid Sequencing

The tethered polymerase of the prior Example, comprising a Bstpolymerase linked to an oligonucleotide tether, was used in an ion-basedsequencing reaction using the Ion Torrent PGM sequencing platform (LifeTechnologies).

Both tethered polymerase and corresponding untethered polymerase(control) were bound to to ssDNA beads and washed with high salt. Thesequencing primer was then hybridized to the ssDNA template. Followinghybridization, the beads were run in a standard sequencing reaction onthe Ion Torrent PGM Sequencing platform. As depicted in FIG. 9 and FIG.10 sequencing using high-salt washes between successive extensions inthe PGM platform was observed to be supported by the tethered polymeraseof Example 1, but not with the corresponding (control) untetheredpolymerase. The use of high salt buffers is advantageous because itincreases polymerase activity.

As depicted in FIG. 9, the Ion Sequencing Polymerase (in untetheredform) was observed to bind Ion Spheres (beads) with ssDNA template inthe Ion W2 reagent (6.3 mM MgCl₂, 13 mM NaCl, 0.01% TritonX-100, pH 7.5)with low affinity and could be washed off with increasing ionicstrength. At 1 M NaCl, 100% of Ion Sequencing Polymerase 1.0 was washedoff Ion Spheres.

As depicted in FIG. 10, the Ion Sequencing Polymerase tethered with anoligo as described in Example 1, where the tether immobilizes thetethered polymerase to the ssDNA template, was observed to remain on theIon beads under high salt conditions as the tethered polymerase was notwashed off in these conditions. High salt is not expected to harm thepolymerase except to wash it from the ssDNA.

The performance of the Ion Sequencing Polymerase in both tethered anduntethered (control) forms in an Ion PGM Sequencing system was measuredand compared.

The untethered form of the Ion Sequencing Polymerase exhibited no signalusing high salt sequencing conditions (20 mM MgCl2, 200 mM NaCl, 0.01%Triton X-100, pH 7.5), and no observable sequencing reads were obtained.

In contrast, the tethered Ion Sequencing Polymerase demonstrated robustsequencing performance using the high salt sequencing conditions (20 mMMgCl2, 200 mM NaCl, 0.01% Triton X-100, pH 7.5), where over 800,000 Q17reads were obtained and over 30 key signals detected from a singlesequencing reaction. As these results indicate, PGM sequencing at highsalt (>200 mM ionic strength) is supported by the tethered IonSequencing Polymerase but not by the untethered Ion SequencingPolymerase.

What is claimed:
 1. A composition comprising an active B st DNApolymerase enzyme, or a mutant or fragment thereof that can catalyzenucleotide incorporation, covalently linked to a first polynucleotide, asecond polynucleotide which is hybridized to the first polynucleotideand a sequencing primer capable of hybridizing to the secondpolynucleotide, wherein the second polynucleotide is a single strandedDNA (ssDNA) template that is covalently linked to a surface and whereinthe composition is present in an aqueous solution with an ionic strengthequal to or greater than 200 mM.
 2. The composition of claim 1, whereinthe first polynucleotide is covalently linked to an amino acid residueof the active Bst DNA polymerase enzyme.
 3. The composition of claim 1,wherein the first polynucleotide is covalently linked to a cysteineresidue of the active Bst DNA polymerase enzyme.
 4. The composition ofclaim 1, further comprising one or more nucleotides.
 5. The compositionof claim 1, wherein the surface further comprises a thirdpolynucleotide.
 6. A composition comprising: an active Bst DNApolymerase enzyme, or a mutant or fragment thereof that can catalyzenucleotide incorporation, having an amino acid residue that iscovalently linked to one end of a first polynucleotide, wherein thefirst polynucleotide is hybridized to a second polynucleotide, whereinthe second polynucleotide is a single stranded DNA (ssDNA) template withone end covalently linked to a surface and wherein the composition ispresent in an aqueous solution of a sequencing by synthesis reactionmixture with an ionic strength equal to or greater than 200 mM.
 7. Thecomposition of claim 6, wherein the amino acid residue comprises acysteine residue of the polymerase.
 8. The composition of claim 6,further comprising one or more nucleotides.
 9. The composition of claim1, wherein the second polynucleotide is covalently attached to thesurface of a bead.
 10. The composition of claim 1, comprising apopulation of identical ssDNA templates covalently linked to thesurface.
 11. The composition of claim 1, wherein the Bst DNA polymeraseis a mutant polymerase comprising one or more mutations.
 12. Thecomposition of claim 6, wherein the second polynucleotide is covalentlyattached to the surface of a bead.
 13. The composition of claim 6,further comprising a population of identical ssDNA templates covalentlylinked to the surface.
 14. The composition of claim 6, wherein the BstDNA polymerase is a mutant polymerase comprising one or more mutations.15. The composition of claim 13, further comprising a sequencing primerhybridized to a ssDNA template.